Two conditions appear essential for fast synchronous neurotransmitter release: First, an optimal adjustment of CA/2+ entry kinetics and second, and optimal assembly of the CA2+ entry site in close proximity to the Ca/2+ entry site in close proximity to the Ca/2+ sensor site to ensure a short diffusion passage for Ca/2+ ions. Our previous genetic analysis of cysteine string protein (Csp) mutations in Drosophila indicates that CSP mediates synchronous neurotransmitter release but not vesicle recycling. In vitro studies of vertebrate CSP suggest a cooperative interaction of CSP with bovine 70kD heat shock cognate protein (HSC70), which is suggested to mediate the uncoating of clathrin-coated vesicles during vesicle recycling Combining the genetic and biochemical evidence, we propose that CSP may cooperatively interact with HSC70 to mediate synchronous release by directing HSC70 to the synchronous release machinery and stimulating its activity. This model postulates a novel function for HSC70 in neurotransmitter release. The focus of this proposal will be to stringently test this hypothesis in vivo by analyzing synchronous neurotransmitter release in mutant Drosophila strains which lack HSC70 and/or CSP function in Drosophila, five distinct constitutively expressed heat shock cognate genes (HSC1-5) have been identified which all share a significant sequence homology with bovine HSC70. However, no fly mutations have been reported for either gene. Since it will be critical for our success to study the correct homologous protein, we propose in Aim 1 to identify the true HSC70 homologue by its biochemical interaction with CSP. To test some of the suggested functions of HSC70., we propose in Aim 2 to determine the in vivo role of HSC70 for synchronous release and for vesicle recycling. Therefore, we will study the effects caused by the loss of HSC70 function in mutant Drosophila. Specifically, we will use electrophsiological recordings and FM1-43 imaging at the larval neuromuscular junction to demonstrate any impairment of neurotransmission. In Aim 3 we propose to test whether CSP and fly HSC70 cooperatively interact in vivo. This will achieved by an analysis of synchronous release and vesicle recycling of double mutant flies which lack simultaneously CSP and HSC70 cooperatively interact in vivo. This will achieved by an analysis of synchronous release and vesicle recycling of double mutant flies which lack simultaneously CSP and HSC70 function. Alternatively, we will determine whether the over expression of HSC70 is able to rescue to neurotransmitter release defect caused by the loss of CSP function. This will also facilitate to determine the hierarchical order of CSP and HSC70 functions in transmitter release. The proposed work may significantly help to understand the molecular mechanism of synchronous neurotransmitter release which is one of the basic conditions mediating the functional plasticity of our nervous system. This understanding will help to fight the dramatic effects of synaptic dysfunction for human life.